Carleton, J. B. | Search bioRxiv

bioRxiv2020

Regulatory Sharing Between Estrogen Receptor α Bound Enhancers

The human genome encodes an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by the combined action of multiple enhancers. We have previously shown that neighboring estrogen-responsive enhancers, which are approximately 5,000 basepairs apart, exhibit complex synergistic contributions to the production of an estrogenic transcriptional response. Here we sought to determine the molecular underpinnings of the observed enhancer cooperativity. We generated genetic deletions of individual estrogen receptor (ER) bound enhancers and found that enhancers containing full estrogen response element (ERE) motifs control ER binding at neighboring sites, while enhancers with pre-existing histone acetylation/accessibility confer a permissible chromatin environment to the neighboring enhancers. Genome engineering revealed that a cluster of two enhancers with half EREs could not compensate for the lack of a full ERE site within the cluster. In contrast, two enhancers with full EREs produced a transcriptional response greater than the wild-type locus. By swapping genomic sequences between enhancers, we found that the genomic location in which a full ERE resides strongly influences enhancer activity. Our results lead to a model in which a full ERE is required for ER recruitment, but the presence of a pre-existing active chromatin environment within an enhancer cluster is also needed in order for estrogen-driven gene regulation to occur.

genetics↗

bioRxiv2018

Sufficiency analysis of estrogen responsive enhancers using synthetic activators

Multiple regulatory regions bound by the same transcription factor have been shown to simultaneously control a single genes expression. However, it remains unclear how these regulatory regions combine to regulate transcription. Here we test the sufficiency of promoter-distal estrogen receptor (ER)-binding sites (ERBS) for activating gene expression by recruiting synthetic activators in the absence of estrogens. Targeting either dCas9-VP16(10x) or dCas9-p300(core) to ERBS induces H3K27ac and activates nearby expression in a manner similar to an estrogen induction, with dCas9-VP16(10x) acting as a stronger activator. The sufficiency of individual ERBS is highly correlated with their necessity, indicating an inherent activation potential that is associated with the binding of RNA polymerase II and several transcription factors. By targeting ERBS combinations, we found that ERBS work independently to control gene expression when bound by synthetic activators. The sufficiency results contrast necessity assays that show synergy between these ERBS, suggesting that synergy occurs between ERBS in terms of activator recruitment, whereas directly recruiting activators leads to independent effects on gene expression.

systems biology↗